The present study investigated the profiles of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) of the aqueous humor (AH) and plasma (PL) in myopia patients, to determine whether there was an association between these levels with their axial length (AL) and to investigate if MMPs/TIMPs were regulated locally or systemically.  A cross-sectional study was conducted. Thirty-nine patients (78 eyes) diagnosed with high myopia were recruited. The AL was measured using IOL Master. And the patients were divided into three groups based on their AL, Group A (AL ≤ 26 mm), Group B (26 < AL ≤ 28 mm), and Group C (AL > 28 mm). The AH in both eyes and blood samples were collected before the patients underwent implantable collamer lens surgery. In all, 78 samples of the AH and 39 samples of the PL were analyzed using MILLIPLEX map assays, followed by statistical analyses of the results. p = 0.014, Β = 0.118), MMP-2 , MMP-9 , and TIMP-1  in the AH were positively associated with the AL. MMP-1  and TIMP-1  concentrations in the PL increased linearly with longer ALs. No concentration-dependent relationship was found between MMP-2 in the PL and AL.  There were 8 patients (16 eyes) in Group A, 22 patients (44 eyes) in Group B, and 9 patients (18 eyes) in Group C. MMP-1 ( There was a consistent relationship between MMP-2 in the AH and AL. AL was not consistently or substantially affected by MMP-2 in the PL, indicating myopia formation was possibly a localized process. Associations among MMP-1, MMP-9, and TIMP-1 in the AH and AL were also observed. The incidence of myopia has been increasing, particularly in East Asia. According to a recent cross-sectional study, the prevalence of myopia in Shanghai University students has reached 92%, with 23% of these students diagnosed with high myopia . High myAlthough several methods such as low concentrations of atropine, outdoor activities, and orthokeratology have been proposed to control myopia progression, their treatment efficacy differs by individual –5. ThereThe progression of myopia is strongly associated with scleral remodeling and excessive AL elongation. The mechanism of scleral remodeling involves the rearrangement of existing material due to microdeformations . Myopic In a previous study, we reported that MMP-2 concentrations in human AH were positively correlated with the AL . Other sIn this study, we examined the MMP and TIMP profiles in the AH and plasma (PL) in patients with myopia but without other systemic diseases, established whether there was a concentration-dependent relationship between patients with different ALs, and investigated whether there was a relationship between MMPs and TIMPs in the PL and AH.39 patients (78 eyes) with high myopia who underwent ICL surgery between January 2018 and October 2018 were recruited. The patient selection criteria included the following: age between 18 to 45 years, spherical equivalent (SE) of both eyes <-6.0 D, AL > 24 mm. The exclusion criteria included patients with contraindications for ICL surgery, a history of cancer, systemic inflammatory diseases (such as rheumatoid arthritis or systemic lupus erythematosus), infectious diseases , uncontrolled hypertension, uncontrolled diabetes, and other systemic chronic diseases. Patients with a history of glaucoma, retinal detachment, or ocular trauma were also excluded. A full inquiry about their recent oral medication, oral supplements, and eye drops being used was conducted.Before surgery, each patient received a comprehensive ocular examination at the Department of Ophthalmology at Shanghai Ninth People's Hospital Affiliated Shanghai Jiao Tong University School of Medicine. Objective refraction was measured using an open-field autorefractor . The AL was measured using a Zeiss IOL Master laser interferometer . According to the AL, the 78 eyes were divided into three groups: Group A (AL < 26 mm), Group B (AL = 26–28 mm), and Group C (AL > 28 mm). Thirty minutes after the patient was given three drops of 1% tropicamide per eye, a dilated fundus exam was performed. The enrolled participants were classified according to the ATN grading system (based on atrophy (A), traction (T), and neovascularization (N)) . One patThis study was approved by the Human Subjects Ethics Subcommittee of the Shanghai Ninth People's Hospital Affiliated Shanghai Jiao Tong University School of Medicine, and the study protocol adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent.g for 10 min at 4°C. The AH and PL were stored in 0.1 mL Eppendorf tubes at -80°C, and the samples were frozen within 4 h of collection.Blood samples were collected in EDTA tubes 10 min before surgery. AH samples were collected at the beginning of surgery. We aspirated the AH from the limbus by anterior chamber paracentesis using a 26-gauge needle using a 1 mL microsyringe. All samples were placed on ice immediately and then transported to the laboratory. Blood samples were transferred to a sterile 2.0 mL Eppendorf tube, then centrifuged at 1000 × All samples were assayed for the total protein levels of MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, TIMP-1, TIMP-2, TIMP-3, and TIMP-4 using the Luminex system  with commercially available MILLIPLEX xMAP Kits . This technology uses multiplexed microsphere-based immunoassays, which apply flow cytometric resolution to spectrally measure distinct microspheres coupled with capture molecules and reporter fluorochromes bound to detection antibodies. The assays were performed following the manufacturer's guidelines and measured using a Luminex 200™ system (Bio-Rad).The Human MMP Panel 2 Multiplex Assay  and the Human TIMP Panel 2 Multiplex Assay  were used. Samples were prepared by following the manufacturer's instructions. The amounts of MMPs/TIMPs (pg/mL) were calculated from the standard curves for each MMP/TIMP according to the manufacturer's instructions.The same AH samples and PL samples were measured in each plate for an interassay control. Interassay coefficients of variation (CVs) and the range of detection are shown in supplementary materials  or sex (p = 0.316). As expected, the ALs and SEs were statistically different (p ≤ 0.001) between the three groups. The SE did not significantly differ between OD and OS (p = 0.566). Among the 39 patients, there were 5 patients with anisometropia (differences in SE ≥ 2.0 D).AH samples were obtained from 78 eyes from 39 patients who underwent ICL surgery. In all, 16 eyes with AL < 26 mm (Group A), 44 eyes with AL = 26–28 mm (Group B), and 18 eyes with AL > 28 mm (Group C) were included. The AL (mean ± SD) was 27.15 ± 1.51 mm in the studied patients, with a mean age of 25.03 ± 6.85 years . The meaThe concentrations of MMPs and TIMPs in AH and PL of all samples are shown in p = 0.002) (p = 0.192) (p = 0.002) and PL (p = 0.016) in Group C  . However= 0.192) . Notably Figures .Moreover, the concentration of TIMP-3 in the AH in Group B was the lowest among the three groups . For thep = 0.014), MMP-2 (p ≤ 0.001), MMP-9 (p ≤ 0.001), and TIMP-1 (p = 0.014) in the AH were correlated with the AL  and TIMP-1 (p = 0.030) in the PL were associated with AL. TIMP-3 might also be associated with the AL (p = 0.050), but statistical significance was not found  and sex (p = 0.180) were not statistically associated with differences in ALs among our patients.According to analyses of the GEE, age  were higher in the AH than in the PL. Second, MMP-1, MMP-2, MMP-9, and TIMP-1 in the AH were positively associated with the AL. In the PL, MMP-1 and TIMP-1 concentrations increased linearly with a longer AL. However, there was no clear concentration-dependent relationship between MMP-2 in the PL and AL. Finally, no significant correlation was found between the AH concentration and the PL concentration of each protein studied.The mechanism of myopia is not completely understood, but AL elongation and scleral remodeling are considered important processes during myopia development . During Previous studies have reported that MMP-2 is elevated in the AH and vitreous humor in myopic eyes , 18; ourIn this study, MMP-1 was positively correlated with AL, both in the AH and PL. MMP-9 in the AH was also positively correlated with AL. MMP-1  degrades type I, II, and III collagens. It has been considered a candidate gene in myopia formation in several studies, but the results have been conflicting –35. In oAs for the association between MMP-9  and myopia, few previous reports were found. However, abnormal expression of MMP-9 has been observed in other ocular diseases. As a key proteolytic enzyme in the degradation of ECM , MMP-9 lTIMPs are the natural inhibitors of MMPs. The activity of MMPs is mainly inhibited by TIMPs via two processes. During enzyme activation, TIMP can form stable complexes with pro-MMP. In the activated MMP phase, TIMP-1 and TIMP-2 can directly form a close complex with activated MMP . AccordiTIMP-2 plays a dual role in regulating MMP-2. Exogenous administration or recombinant expression of TIMP-2 resulted in inhibition of MMP-2 activation, while endogenous physiological upregulation of TIMP-2 promoted MMP-2 activation . Kudo etMoreover, we observed higher concentrations of TIMP-1, TIMP-2, and TIMP-3 (OS) in the AH than in the PL. It suggests that they could be actively transported in the AH, and/or synthesized locally. Notably, we found that the concentrations of TIMP-3 were higher in the left eyes than in the right eyes. The levels of TIMPs were measured at the same time in the same manner for each sample, and no difference was found in the levels of TIMP-1, TIMP-2, and TIMP-4. Besides, only 5 patients (12.8%) had anisometropia (SE ≥ 2.0 D). Among the participants, no significant difference was found between the SE of both eyes. One possible explanation is that we collected AH first from the OD. After completing the ICL procedure for the OD, we started collecting the AH from the OS. There was an interval of 15 min between the two eyes of the same patient. Although the underlying mechanism was not understood, it is estimated that TIMP-3 in the OS increases rapidly when the OD is being operated on. By changing the sequence of the eyes to operate, future study might be able to elucidate.To the best of our knowledge, our study is the first to demonstrate a positive correlation between concentrations of MMP-9 in the AH and AL. And we found a positive correlation between concentrations of MMP-1 and TIMP-1 in the PL and AL. A strength of this study was that we enrolled patients at a young age, so that we could minimize the influence from other age-related systemic diseases. We also did not recruit cataract patients in the control group, AH of such eyes present with increased malondialdehyde and lipid peroxide, which may affect the levels of MMPs/TIMPs.The limitations of our study should be discussed. First, the small number of subjects limited our results. Further studies enrolling a larger number of patients and more patients with anisometropia are needed. Second, we conducted a cross-sectional study reflecting temporal relationships between AL and MMPs/TIMPs. All patients were adults and were in the stationary stage of myopia. Therefore, we could not observe the changes of MMPs/TIMPs during myopia formation. Thirdly, our study did not have a control group with normal axial length. Anterior chamber paracentesis is an invasive procedure that raises ethical concerns if we include normal young subjects. However, age-related cataract patients with normal axial length have a large difference between our studied population in age. The possible impact of systemic diseases, including dyslipidemia and diabetes mellitus, on the plasma MMP/TIMP profile also makes cataract patients less suitable as control. Thus, we decided to compare between the groups with different axial lengths in order to minimize other influences. Correlation analyses resulted in new insights into myopia formation. The concentration of MMP-2 in the PL did not correlate with the AL. We hypothesize that in patients without systemic disease, it is possible that the changes of MMP-2 could be induced locally. When the blood-aqueous barrier is intact, the potential myopia-inducing factors including MMPs and TIMPs could be more likely to be regulated within the ocular system. Other studies have provided some evidence for this possibility. For example, optic nerve blockade does not prevent myopia induced by form deprivation , and resNevertheless, some case reports have demonstrated that myopia can be induced by systemic diseases, such as SLE  and famiIn conclusion, we characterized MMP/TIMP profiles in the AH and PL in myopia patients. We found a positive correlation between AL and levels of MMP-1 and TIMP-1, both in the AH and PL, despite a rather low concentration of MMP-1 in the AH. A positive correlation between AL and levels of MMP-9 in the AH was also observed. Our study showed a consistent relationship between MMP-2 in the AH and AL. However, the AL was not consistently or substantially affected by MMP-2 in the PL, leading to the hypothesis that abnormal expression of MMP-2 in the AH, which is closely related to scleral remodeling, could more likely be induced locally, rather than systematically. Additional studies about the role of MMP-1 and MMP-9, the activity of MMP-2, and the homeostasis of pro-MMP-2 and TIMPs are needed, to validate these preliminary results.


The possible target genes (RNF24 and SIGLEC) were further screened by reverse transcription-quantitative PCR. In addition, functional experiments showed that miR-3913-5p might be an oncogene that affects the proliferation and migration of CCA cells by inhibiting and mimicking miR-3913-5p. Therefore, miR-3913 may serve as a biomarker for the diagnosis and prognosis of patients with CCA.Accumulating evidence has demonstrated that microRNAs (miRNAs or miRs) play an important role in the diagnosis and prognosis of tumors. In the case of cholangiocarcinoma (CCA), miRNAs may serve as potential tumor biomarkers and therapeutic targets. Based on The Cancer Genome Atlas (TCGA) database, fold change >2 was used to screen out miRNAs with differential expression in patients with CCA. Univariate and multivariate Cox regression analyses identified miR-3913-5p as an independent prognostic factor in patients with CCA. Overall survival and progression-free survival of patients with CCA were analyzed based on clinical data from TCGA database. In addition, four datasets were combined to identify 21 possible target genes of miR-3913, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to predict potential pathways and functions of the molecular target genes. Subsequently, the miRNAs associated with survival were selected to build the miRNA-mRNA expression network. Furthermore, the differential expression of miR-3913-5p in CCA cells and normal bile duct epithelial cells was confirmed through  Cholangiocarcinoma (CCA) is the second most common malignancy of the hepatobiliary system, second only to hepatocellular carcinoma, and the incidence of CCA has been increasing in the past 40 years , 2. Due MicroRNAs (miRNAs) are small single-stranded RNAs that measure ~22 nt and are processed from intracellular transcripts of hairpin structures. The processing and maturation of miRNAs undergo a spatial transition from the nucleus to the cytoplasm, and the whole process is coordinated by a variety of enzymes and helper proteins subjected to multilevel regulation and multistep precise reactions . miRNAs in vitro experiments. The aim of this study was to develop CCA biomarkers to predict the survival and prognosis of patients with CCA based on miR-3913-5p.However, the role of miR-3913-5p in tumors has not been reported. The present study identified high expression of miR-3913 in CCA through The Cancer Genome Atlas (TCGA) database. The role of miR-3913 in the proliferation and migration of CCA has been demonstrated for the first time by bioinformatic analysis and https://portal.gdc.cancer.gov/) according to the following inclusion criteria: (1) samples contained complete miRNA sequences and clinical information and (2) samples had prognostic information. Patients were excluded if they (1) lacked miRNA sequencing data, (2) did not have prognostic information, (3) were not primary cholangiocarcinoma, and (4) were missing clinical data. Until December 20, 2019, a total of 45 patients (36 patients with CCA and 9 benign controls) had been included.miRNA sequences and clinical information were downloaded from TCGA database (Two independent datasets downloaded from the Gene Expression Omnibus (GEO) database were used to verify the miRNA-3913-5p expression in patients with CCA (GSE85589 and GSE53992).P < 0.05 was considered to indicate a statistically significant difference.PCA is a mathematical algorithm that can reduce data dimensions while retaining the vast majority of variables in a dataset . The appP < 0.05 were considered to exhibit a statistically significant difference in terms of survival and therefore were included in further analyses.The R package “survminer” was utilized to determine the cut-off point for patient stratification into two groups. Overall survival (OS) and progression-free survival (PFS) were assessed in the high- and low-expression groups by Kaplan-Meier survival analysis (log-rank method). Those miRNAs with Tumor incidence from TCGA database was included as an outcome indicator, and the R package “pROC” was utilized to construct receiver operating characteristic (ROC) curves for evaluating the diagnostic model. Similarly, the survival status and survival time of the samples were extracted, and the R package “timeROC” was used to evaluate the accuracy of mir-3913-5p as a prognostic biomarker.http://www.mirdb.org/), miRWalk , TargetScan (http://www.targetscan.org/), and miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/index.php) were used to predict the target genes of miRNA, and the intersection of these four online tools was selected by Venn diagram to further screen out more reliable target genes. Subsequently, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to predict the functional enrichment and functional pathways of miRNA target genes. In addition, Gene-Set Enrichment Analysis (GSEA) was performed to further supplement the results of GO and KEGG analyses of miRNA target genes.The online tools miRDB   was used as complete medium for the above cells, which were cultured at 37°C in humid air under 5% COMiR-3913-5p mimics , inhibitors , negative control (NC), and inhibitor negative control (INC) were purchased from Guangzhou RiboBio Co., Ltd. Lipofectamine 3000  was used as a transfection carrier. The transfection efficiency was validated by reverse transcription-quantitative PCR (RT-qPCR) analysis.ΔΔCq- method. The primers for U6, miR-3913-5p, and target genes were designed and synthesized by Sunya. The following primer sequences were used: U6, forward (F) 5′-CTCGCTTCGGCAGCACA-3′ and reverse (R) 5′-AACGCTTCACGAATTTGCGT-3′; and miR-3913-5p, F 5′-CGCGCGTTTGGGACTGATCTTG-3′ and R 5′-AGTGCAGGGTCCGAGGTATT-3′. The sequences of the primers for the target genes are shown in Table Total RNA was extracted from the cell lines  using TRIzol reagent . The extracted RNA was reverse transcribed using the PrimeScript™RT reagent Kit with gDNA Eraser , and the expression level of miR-3919-5p was measured with the SYBR Green PCR Master Mix kit . U6 served as an internal reference to measure the relative miRNA expression by the 2μl CCK-8 solution  was added to each well and incubated for 2 h in a 5% CO2 incubator at 37°C. Finally, the absorbance at 450 nm was determined using the SpectraMax Absorbance Reader CMax Plus  at 0, 24, 48, 72, and 96 h. Each experiment was repeated ≥3 times.The CCA cell lines (HCCC-9810 and RBE) were transfected with miR-3913-5p mimics, miR-3913-5p inhibitors, NC, or INC and then seeded in 96-well plates with 3,000 cells per well. A total of 10 2 incubator at 37°C for 14 days. Cells were photographed, and the number of colonies with >50 μm diameter was calculated. The experiment was repeated ≥3 times.The transfected cells were seeded in 6-well plates with 1,000 cells per well and then cultured in a 5% COμm inserts were purchased from EMD Millipore. For the migration assay, the HCCC and RBE cell lines (1 × 105 cells per well) were added to the upper chamber in the presence of serum-free RPMI-1640 medium. A total of 500 μl complete medium (RPMI − 1640 + 10%FBS) was added to the lower chamber. The 24-well plates were placed in a cell incubator and incubated at 37°C and 5% CO2 for 48 h. Next, cells in the upper compartment that failed to pass through the spoon were removed and washed with PBS 2-3 times. The cells that passed through the upper compartment and adhered to its lower surface were fixed with 4% tissue fixation solution  for 0.5 h, stained with 0.1% crystal violet for 0.5 h, air dried at room temperature, and observed under the OLYMPUS BX43 (Olympus Corporation). Five fields of view were randomly selected for counting . The assay was carried out 3 times independently.Twenty-four-well cell culture plates with 8 t-test was used to compare the differences between two groups. P < 0.05 was considered to indicate a statistically significant difference.The SPSS 20.0 software (IBM Corp.) was used for statistical analysis. Comparison between cell lines was done using one-way ANOVA test followed by post hoc test (Dunnett). The log-rank test was used to compare the survival of the high- and low-expression groups. Univariate/multivariate Cox multivariate hazard regression analyses were used to screen for survival-associated miRNAs and independent factors affecting prognosis based on clinical information. Two-way ANOVA test (Sidak's multiple comparisons test) was used to compare the cell proliferation. The Student's Through PCA analysis, a PCA diagram was drawn, and similarities and differences were found between samples . AccordiP < 0.05). To further screen out miRNAs that independently affect survival, the above 3 miRNAs that may be correlated with survival were included in the multivariate Cox regression analysis, and the results showed that miR-3913-5p may be an independent factor influencing the survival time of patients with CCA  (P = 0.0071)  and miRNA expression data using the “survminer” package . The res 0.0044)  and that 0.0071) . In addi 0.0071) . It shou 0.0071) . The accThe intersection of the prediction results of target genes from the four databases  revealed 21 reliable candidate target genes . SubsequFirst, inhibitors of miR-3913-5p were transfected into HCCC and RBE cells, and successful transfection was confirmed by RT-qPCR Figures . Then, CCholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumors that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for 15% of all primary liver cancers and 3% of gastrointestinal malignancies . Dysreguin vitro experiments. A diagnostic and prognostic model of miR-3913-5p was constructed for the first time, which improved the diagnostic effectiveness of CCA to a certain extent, and it is beneficial to observe the prognosis of patients with CCA as a biomarker. However, due to the insufficient sample size, this novel biomarker may not be reliable and needs to be further confirmed by including a larger sample size or by using multiple miRNAs combined diagnostic models. Previous studies have confirmed that this diagnostic panel with multiple miRNAs can effectively improve the sensitivity of diagnosis, but an excessive number of molecules should not be included to avoid reducing the specificity of diagnosis [The present study was the first to report the carcinogenic role of miR-3913-5p in tumors, especially in CCA. Samples of the CCA and control groups were extracted based on TCGA database and verified by the GEO database and iagnosis , 33. In To further explore the molecular mechanism of miR-3913-5p, the present study focused on the prediction of target genes of miR-3913-5p and further explored the function of target genes through GO, KEGG, and GSEA analyses. Since the majority of the target genes were associated with negative regulation of cell metabolism, it was shown that miR-3913-5p might be a potential tumor promotor. Furthermore, according to the two candidate target genes SIGLEC10 and RNF24, it was predicted that SIGLEC10 might affect the metastasis of CCA by changing the cell adhesion ability through the above analysis. Some studies have confirmed that the combination of vascular adhesion protein-1 and SIGLEC10 could affect cell adhesion . RNF24 min vitro experiments. Due to the complexity of the body, in vivo experiments are needed to further confirm the role of miR-3913-5p in the progression of CCA.However, the present study has several limitations. First, the sample size of TCGA was too small, the diagnostic panel was relatively unstable, and the prediction might be biased. Second, the sequencing results were obtained from the CCA tissues and the corresponding control group which may not accurately reflect the levels of miRNAs in saliva, serum, urine, or stool. Hence, we need to further explore the expression of miR-3913-5p in the above samples to facilitate the detection of its expression level. In addition, the experimental validation only included In conclusion, we have demonstrated that miR-3913-5p is upregulated in CCA cell lines, and high expression of this gene is associated with poor prognosis based on TCGA database. Especially, it has been served as an independent factor of the prognosis of CCA according to the univariate and multivariate analyses. Furthermore, we have revealed for the first time the role of this oncogene in promoting the proliferation and migration of CCA cells, and its potential target genes are SIGLEC10 and RNF24. Taken together, these findings describe a novel and promising biomarker for prognosis in CCA.


We performed a trial-based cost-effectiveness analysis of a lifestyle intervention targeted at preventing diabetes in India.Data on the cost-effectiveness of lifestyle-based diabetes prevention programs are mostly from high-income countries, which cannot be extrapolated to low- and middle-income countriesThe Kerala Diabetes Prevention Program was a cluster-randomized controlled trial of 1007 individuals conducted in 60 polling areas  in Kerala state. Participants (30–60 years) were those with a high diabetes risk score and without diabetes on an oral glucose tolerance test. The intervention group received a 12-month peer-support lifestyle intervention involving 15 group sessions delivered in community settings by trained lay peer leaders. There were also linked community activities to sustain behavior change. The control group received a booklet on lifestyle change. Costs were estimated from the health system and societal perspectives, with 2018 as the reference year. Effectiveness was measured in terms of the number of diabetes cases prevented and quality-adjusted life years (QALYs). Three times India’s gross domestic product per capita (US$6108) was used as the cost-effectiveness threshold. The analyses were conducted with a 2-year time horizon. Costs and effects were discounted at 3% per annum. One-way and multi-way sensitivity analyses were performed.Baseline characteristics were similar in the two study groups. Over 2 years, the intervention resulted in an incremental health system cost of US$2.0 , incremental societal cost of US$6.2 , absolute risk reduction of 2.1%, and incremental QALYs of 0.04 per person. From a health system perspective, the cost per diabetes case prevented was US$95.2, and the cost per QALY gained was US$50.0. From a societal perspective, the corresponding figures were US$295.1 and US$155.0. For the number of diabetes cases prevented, the probability for the intervention to be cost-effective was 84.0% and 83.1% from the health system and societal perspectives, respectively. The corresponding figures for QALY gained were 99.1% and 97.8%. The results were robust to discounting and sensitivity analyses.A community-based peer-support lifestyle intervention was cost-effective in individuals at high risk of developing diabetes in India over 2 years.ACTRN12611000262909). Registered 10 March 2011.The trial was registered with Australia and New Zealand Clinical Trials Registry ( Type 2 diabetes is a major cause of death and disability worldwide, with its health burden falling increasingly upon the low- and middle-income countries (LMICs) . India hEvidence from randomized controlled trials (RCTs) shows that lifestyle interventions for individuals at high risk of developing type 2 diabetes can reduce progression to diabetes , microvaOnly one previous study, the Indian Diabetes Prevention Programme (IDPP), has undertaken a cost-effectiveness analysis of lifestyle intervention among high-risk individuals for developing diabetes in India. The IDPP estimated a cost of US$1052 to prevent one case of diabetes over 3 years . For exaWe conducted a cluster-RCT of a community-based peer-support lifestyle intervention among individuals at high risk of developing type 2 diabetes in the Indian state of Kerala, the Kerala Diabetes Prevention Program (K-DPP) , 15. AftDetails of the K-DPP study design have been previously published . BrieflyThe development and cultural adaptation of the intervention program have been explained in detail elsewhere . BrieflyWe estimated costs from the health system and societal perspectives, in accordance with the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) statement  and costTo estimate health system costs, we considered only direct medical costs, which included screening costs, intervention costs, and healthcare utilization costs.Most of our screening costs were driven by the need to identify high-risk individuals for the trial in a short period of time through home visits and community-based clinics , 26. FroInformation on healthcare use  from public and private facilities was collected annually based on self-reports or from participants’ prescriptions, medical records, and bills, if available. In Kerala, the majority of outpatient (63.5%) and inpatient care (66%) are sought from private facilities . PrivateTo estimate societal costs, we considered direct medical costs, direct non-medical costs, and indirect costs. Direct non-medical costs included expenses incurred by participants for transport, food, and accommodation while seeking healthcare (self-reported), and for the time spent traveling to and attending group sessions. The intervention participants (including peer leaders) spent, on average, 30 min of travel time and 60 min in attending one group session. These times were valued at the minimum hourly wage of an unskilled worker employed in the agricultural sector in India . Indirecp = 0.405)) at 2 years. Our trial was likely underpowered to detect a significant difference, given the inclusion of participants with normal plasma glucose  [We assessed effectiveness in terms of the number of diabetes cases prevented and of QALYs, as recommended by the CHEERS statement  and costriteria) , and theriteria) , Briggs riteria) . This isriteria) , 25 and riteria) –40.QALYs were estimated based on the utility values derived from the 36-item Short Form (SF-36) health survey . SF-36 wThis cost-effectiveness analysis was conducted using standard methods of trial-based economic evaluation . The anaWe estimated uncertainty in the estimates of costs and effects by non-parametric bootstrapping method, which was conducted by resampling without replenishment from the original dataset . In eachAdditionally, we conducted the following one-way and multi-way sensitivity analyses. We allowed the cost of group sessions (the key driver of intervention costs) and effects to vary by a range of plausible values  in a non-research setting, based on assumptions made in previous lifestyle-based diabetes prevention programs , 48. ThiA total of 1007 participants (500 in the intervention group and 507 in the control group) were recruited into the trial. All 60 clusters and 964 (95.7%) participants (96.4% in the intervention group and 95.1% in the control group) were followed up at 2 years. The baseline characteristics of clusters and participants were similar in the two study groups , resulting in an NNT of 47.6. The average QALYs gained were consistently higher in the intervention group compared to the control group in both the first (0.81 vs. 0.79) and second years (0.84 vs. 0.81). Overall, during the 2-year trial period, participants in the intervention group accrued more QALYs than the control participants (1.65 vs. 1.61) with a mean difference of 0.04 .At 2 years, the absolute risk reduction in diabetes incidence was 2.1%  and 100% (for QALYs gained) of bootstrap estimates were cost-effective (<US$6108) from both the perspectives. In addition, the probability for the intervention to be cost-effective was more than 83% for diabetes cases prevented and 97% for QALYs gained from both the perspectives. Discounting costs and effects at a rate of 3% per annum did not alter the results appreciably. In all case scenarios in the sensitivity analyses for both the effects, the ICERs remained below the cost-effectiveness threshold .In our study, the difference in intervention costs between study groups (US$23.4) was accompanied by lower  outpatient, medication, and indirect costs in the intervention group, resulting in a negligible cost difference between study groups from the health system and societal perspectives. This could be due to the positive intervention effects on healthy lifestyle behaviors including physical activity, diet, tobacco use, and alcohol use , 17. In While the NNT to prevent one case of diabetes in our study (47.6) was larger than that reported in the IDPP (6.4)  and US DOur study has several strengths. To our knowledge, this is the first study from an LMIC setting to provide comprehensive evidence on the cost-effectiveness of lifestyle intervention in high-risk individuals for type 2 diabetes from both the health system and societal perspectives. The K-DPP trial was conducted in the Indian state of Kerala, which has the highest prevalence of diabetes in India with a high burden of several cardiovascular risk factors , 56–58. Our study has some limitations. These include, the relatively short follow-up of 2 years, which did not permit an assessment of the long-term cost-effectiveness of the intervention. However, by adopting a 2-year time horizon, we likely underestimated the effects, with the health benefits of a lifestyle intervention generally occurring over the longer term . AnotherIn this trial-based economic analysis, we have demonstrated that implementing a low-cost peer-support lifestyle intervention in community settings for individuals at high risk of developing diabetes was cost-effective in a low- and middle-income setting. Future work is needed to determine whether this persists over a longer period of time and when the intervention model is taken to scale. A recent scale-up of K-DPP to more than 15,000 trained peer leaders who delivered the program to more than 370,000 people in Kerala has demonstrated very promising results . The finAdditional file 1: Table S1. Unit costs used for calculating direct and indirect costs. Table S2. Baseline characteristics of clusters and participants.


Furthermore, we evaluated the properties of the obtained bio-inks to gauge their suitability in bio-printing, primarily focusing on their viscosity, printability, and shrinkage properties. Furthermore, the bio-ink encapsulating the cells (NIH3T3 fibroblast cell line) was characterized using a live/dead assay and WST-1 to assess the biocompatibility. It was inferred from the results that the blended hydrogel was successfully printed for all groups with viscosities of 883 Pa∙s , 1211 Pa∙s , and 1525 Pa∙s,  at a 0.1 s−1 shear rate. Their structures exhibited no significant shrinkage after CaCl2 crosslinking and maintained their integrity during the culture periods. The relative proliferation rate of the encapsulated cells in the HA/SA blended bio-ink was 70% higher than the SA-only bio-ink after the fourth day. These results suggest that the 3D printable HA/SA hydrogel could be used as the bio-ink for tissue engineering applications.Bio-ink properties have been extensively studied for use in the three-dimensional (3D) bio-printing process for tissue engineering applications. In this study, we developed a method to synthesize bio-ink using hyaluronic acid (HA) and sodium alginate (SA) without employing the chemical crosslinking agents of HA to 30% ( Three-dimensional 3D) printing techniques have been usually applied in the field of tissue engineering to fabricate a predesigned scaffold, regenerating a defected tissue in brief time ,2. In paD printinIn this study, we investigated a biologically enhanced bio-ink using HA, which could improve the cellular functionality of the bio-ink using the alginate alone for 3D bio-printing. We anticipate that SA can support the printability of the bio-ink, while HA enhances the bioactivity in the encapsulated cells. The mechanical properties, such as viscosity, printability, and biocompatibility of the 3D bio-printed scaffolds based on the prepared HA/SA blended bio-ink were analyzed.w/v) in a final concentration of 0%, 10% and 30% (w/v), respectively. The group names and compositions of the hydrogels are shown in 2. The cell encapsulated bio-ink gently mixed with HA/SA hydrogels to reach a final concentration of 1 × 106 cells/mL.SA was purchased from Sigma-Aldrich . HA of 2500 kDa was supplied from Humedix.Co. . HA and alginate were dissolved in high glucose-DMEM  with 1% penicillin/streptomycin and physically mixed in different ratio . The shrinking rate analysis of structure area was carried out using an image J program . HA/SA hydrogels were analyzed by the rheometer  at the room temperature to measure the viscosities of the hydrogels. The infrared spectra of HA/SA hydrogel were measured using an FT-IR spectrophotometer  measured spectrum condition at a resolution of 4.0 cm2 size and then analyzed with WST-1  to analyze the cell proliferation for 4 days. Briefly, the constructs were washed with PBS, moved to another 48 well plate. In the next, 40 μL of WST-1 reagent and 400 μL of serum free medium were added in each well and then incubated in the dark for 40 min at 37℃. After incubation, 100 μL of the incubated medium was transferred to a 96 well plate, and the absorbance at 450 nm was immediately measured using a microplate spectrophotometer . The cell viability was examined after incubation for five samples per each group, and the WST-1 reagent with serum free medium was used as the blank control.For cell viability analysis, the encapsulated cells in the constructs were evaluated using a Live/Dead Cell assay kit  according to the manufacturer’s instructions for 7 days. All the constructs were washed with PBS , moved to another 24 well plate. The samples were stained for 20 min in the dark and washed 3 times in PBS. Live and dead cells were observed using fluorescence microscopy . Furthermore, the cell-laden constructs were cut on 2.4 × 2.4 mmp < 0.05. All experiments were carried out at least three times and one-way analysis of variance was conducted for statistical analyses at the significance level of The SA is extensively used as the basement material of the bio-ink in the tissue engineering field because their physical and mechanical properties satisfy the requirements of an ‘ideal’ bio-ink . As show−1 in S100H0, which shifted with adding HA at 3246.46 cm−1 in S70H30. The alkane –CH stretching vibrations were measured at 2883.43 cm−1 in S100H0, which also shifted with adding HA at 2927.59 cm−1 in S70H30. The asymmetric and symmetric stretching of the carboxyl group of SA at 1605.26 and 1404.79 cm−1 in S100H0 were shifted with adding HA at 1598.44 and 1408.80 cm−1 in S70H30, respectively [To characterize the chemistry of the HA/SA blends, we analyzed the pre-crosslinked hydrogels by using FT-IR. As shown in ectively ,21. Thes2 was used for appropriate printing, and then, the scaffolds successfully fabricated with stacked layer-by-layer without any crosslinking of the HA. Even though the size of the strands increased compared to SA-only bio-ink, the scaffolds maintained their porous structure after stacking process. These results indicated the pre-crosslinking rate dependent on the ratio of SA has an important role to construct the strut in the scaffold in contrast to the viscosity results. Furthermore, as shown in 2 was carried out for fully cross-linked SA to stable structure, which could prevent collapse entire structure in early time. One of the major challenges for successful tissue regeneration, the scaffold must maintain the structure during application periods [The scaffolds were printed after preparation and characterization of the bio-ink. In  periods . AlthougCell viability and the cell proliferation of the scaffolds using Live/Dead staining and WST-1 assay, respectively. As shown in Furthermore, in 2, which also well-blended physically according to the ratio of the hydrogels. In particular, the cell viability and proliferation increased with adding the ratio HA in the bio-ink although that were more disintegrated than SA-only scaffold. Furthermore, we could improve the bio-ink properties by applying the motif to enhance the binding affinity with cells on SA hydrogel and increasing the crosslinking property of HA to enhance the cell viability as well as the structural stability. Consequently, we suggest that these blended 3D printable HA/SA hydrogel potentially could be a good candidate for the application of the soft tissue regeneration. In this study, we developed the HA/SA blended hydrogel bio-ink for 3D bio-printing application. The viscosity of the bio-inks was regulated HA with pre-crosslinking treatment using CaCl


Ascaris lumbricoides. Through large-scale deworming programs, World Health Organization aims to reduce morbidity, caused by moderate-to-heavy intensity infections, below 2%. In order to monitor these control programs, stool samples are examined microscopically for the presence of worm eggs. This procedure requires well-trained personnel and is known to show variability between different operators interpreting the slides. We have investigated whether ABA-1, one of the excretory-secretory products of A. lumbricoides can be used as a coproantigen marker for infection with this parasite. Polyclonal antibodies were generated and a coproantigen ELISA was developed. Using this ELISA, it was found that ABA-1 in stool detected Ascaris infection with a sensitivity of 91.5% and a specificity of 95.3%. Our results also demonstrate that there is a correlation between ABA-1 levels in stool and A. lumbricoides DNA detected in stool. Using a threshold of 18.2 ng/g stool the ABA-1 ELISA correctly assigned 68.4% of infected individuals to the moderate-to-heavy intensity infection group, with a specificity of 97.1%. Furthermore, the levels of ABA-1 in stool were shown to rapidly and strongly decrease upon administration of a standard anthelminthic treatment . In an Ascaris suum infection model in pigs, it was found that ABA-1 remained undetectable until day 28 and was detected at day 42 or 56, concurrent with the appearance of worm eggs in the stool. This report demonstrates that ABA-1 can be considered an Ascaris -specific coproantigen marker that can be used to monitor infection intensity. It also opens the path for development of point-of-care immunoassay-based tests to determine A. lumbricoides infection in stool at the sample collection site.Intestinal worms, or soil-transmitted helminths (STHs), affect hundreds of millions of people in all tropical and subtropical regions of the world. The most prevalent STH is  Ascaris lumbricoides is the most prevalent and efforts are ongoing to use preventive chemotherapy to reduce both prevalence and intensity of this infection. To monitor these programs, stool-based microscopy is currently used. We have investigated the possibility of using ABA-1, an abundantly secreted protein from the worm, as a biomarker in stool of infected individuals. We have developed an ELISA and using this assay determined that ABA-1 as stool biomarker had a sensitivity of 91.5% and a specificity of 95.3% to detect infection with A. lumbricoides. We also showed that ABA-1 in stool rapidly and strongly decreased upon administration of a standard anthelminthic treatment. The main asset of this novel stool biomarker is its potential to be used in point-of-care immunoassay-based tests to determine A. lumbricoides infection in stool at the sample collection site.Intestinal worms are one of the most common infections in tropical and subtropical parts of the world. The roundworm  Ascaris lumbricoides, Trichuris trichiura, and the hookworm species Ancylostoma duodenale and Necator americanus. They pose a major threat to public health in large parts of the world. Children and women of childbearing age are at highest risk of developing morbidity, which is mainly associated with moderate-to-heavy intensity (M&HI) infections [According to the World Health Organization (WHO), approximately 1.5 billion people, or 24% of the world’s population, are infected with soil-transmitted helminths (STH) worldwide [fections . To reacMonitoring of the current programs and decision-taking is currently based on the detection and quantification of STH eggs in a stool smear using a compound microscope, the so called Kato-Katz thick smear technique –8. This Fasciola species, Strongyloides stercoralis and Taenia solium [Toxocara species and no specific proteins have been identified that are used as coproantigen for Ascaris species, such as A. suum in pigs and A. lumbricoides in humans [In the veterinary world, the use of coproantigen detection to diagnose helminth infection has become widely adopted –11. Diaga solium –14. To on humans .A. suum [Ascaris worms and some larval stages, could be detected in fecal samples using a coproantigen ELISA [Ascaris [A few studies have explored the excretory-secretory products from different stages of A. suum –18. Thesen ELISA . ABA-1 i[Ascaris –22.Human samples from Kenya were collected as part of a field study in Kenya. The study was approved by the KEMRI Scientific and Ethics Review Unit (SERU), Nairobi, Kenya (Protocol # KEMRI/SERU/CGHR/102/3554). Since all study participants were minors, informed consent forms were signed by parents/guardians of the study participants, and verbal assents were obtained from all study participants.All animal experiments were conducted in accordance with the E.U. Animal Welfare Directives and VICH Guidelines for Good Clinical Practice. Ethical approval to conduct the studies was obtained from the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University.S. mansoni prevalence area) and Siaya county . Stool samples were collected in order to determine the STH and Schistosoma mansoni infection status, based on qPCR-based quantification of helminth DNA present in stool. A total of 474 participants that donated stool samples were included in this study. Of these participants, 71 were found to be positive for A. lumbricoides, 30 for T. trichiura, 16 for hookworm, and 82 for S. mansoni, based on qPCR detection of helminth DNA in stool (see below). After the cross-sectional collection of samples, a total of 22 A. lumbricoides infected individuals were treated with a single dose of albendazole (400mg) and stool samples were collected at day 6, day 12 and day 24 post-treatment. Infection status at each timepoint was determined by qPCR-based quantification of helminth DNA present in stool.Plasma and urine samples from Kenya were collected as part of a field study. The study was approved by the KEMRI Scientific and Ethics Review Unit (SERU), Nairobi, Kenya (Protocol # KEMRI/SERU/CGHR/102/3554). Since all study participants were minors, signed informed consent forms were obtained from their parents/guardians, and verbal assents were obtained from all study participants. This study was undertaken in the former Nyanza province, in the southwest part of Kenya, with collections in the Kisumu county  between egg count and qPCR analysis, qPCR was repeated on a second stool aliquot from the same individual and qPCR results were confirmed. It was therefore decided to use the qPCR data as the reference data set.Helminth infection in humans was assessed on stool samples using the Kato-Katz procedure and/or qPCR analysis, as described before , 25. BriA. lumbricoides  and Kato-Katz data (in epg). Based on this analysis, a qPCR result of 700 cps/rxn was found to correspond to 5,000 epg, i.e. the boundary for moderate infection intensity for ricoides .Stool sample aliqouts—frozen at -80C - were allowed to thaw at room temperature and an aliquot of approx. 250 mg was transferred to a Powerbead tube . Samples were further processed by addition of 1 mL of ice-cold PBS followed by bead beating in a tissue homogenizer  for 1 minute at 6,500 rpm. Stool extracts were centrifuged at 23,000 x g for 15 minutes at 4°C and supernatant was transferred to a protein LoBind tube  and stored at -20°C till further use. Where indicated the bead beating step was omitted from the procedure.A. suum worms were collected from naturally infected pigs at the local slaughterhouse. Collected adult worms were washed three times in tap water and the pseudocoelomic fluid was collected after cutting of the posterior tip off the worm. The pseudocoelomic fluid was then centrifuged for 15 min at 10,000 × g and 4°C and the supernatant filtered (0.22 μm) and stored at -80°C until use. Adult worm protein extracts were produced as described before [Adult d before . BrieflyAn expression vector with the ABA-1 sequence (GenBank ID: AAD13651.1) was constructed and used to produce recombinant his-tagged ABA-1 in a bacterial expression system . The produced recombinant protein was purified using chromatography His-tag based column under denaturing conditions. After elution, to remove urea and imidazole, the purified fraction was dialyzed at 4°C against a refolding buffer containing decreasing concentrations of urea and finally against PBS buffer. Approximately 13 mg of the purified His-tagged protein was obtained and this purified protein was found by SDS-page to have a molecular weight of approximately 32 kDa, similar to the theoretical Mw . A totalFor the preparation of the polyclonal antibody, rabbits were immunized using 4 different injections with 100 μg of the recombinant ABA-1 with a non-Freund adjuvant, based on the Speedy 28-day program . Antibodies were purified from 70 mL serum of the animal with the highest titer using an antigen-specific affinity purification. A total of 24.3 mg of affinity purified IgG was dissolved in 6.2 mL of PBS with 0.01% thimerosal and 0.1% BSA. Aliquots of the purified antibodies, 1:2 diluted with glycerol were stored at -20°C.4. A total of 5 mg of antibody coupled to HRP was obtained in a volume of 2 mL PBS. Aliquots of the HRP conjugated antibodies, 1:2 diluted with glycerol were stored at -20°C.HRP conjugated polyclonal antibody was produced  by covalently conjugating Horse Radish Peroxidase  using NaIOA stock solution of the recombinant ABA-1 was prepared at a concentration of 5 μg/mL in PBS. Calibration samples were generated by first diluting this stock solution in block buffer (Superblock + 0.05% Tween) to a concentration of 100 ng/mL and then 1/3 serial dilutions were generated in block buffer till a concentration of 0.137 ng/mL. Also, a blank sample containing only block buffer was included.The ELISA for coproantigen detection was performed as follows. Flat bottomed polystyrene plates  were coated with purified rabbit anti-ABA-1 IgG diluted to 0.8 μg/mL in PBS and incubated for 2 hours at room temperature. Next, after washing with PBS-T (PBS with 0.05% Tween-20) the plate was blocked with block buffer (Superblock + 0.05% Tween) for 1 hour at room temperature. Calibration samples or stool extracts, diluted 5-fold in block buffer, were added and incubated for 1 hour at room temperature. Subsequently, plates were washed 3 times with PBS-T and purified rabbit anti-ABA-1 IgG conjugated with HRP was added at a concentration of 2.5 μg/mL and incubated for 1 hour at room temperature. Plates were washed 5 times with PBS-T and color development was done using Sureblue TMB . The reaction was stopped after 10 minutes using 1N HCl and optical density was measured at 450 nm.Calibration curves were analyzed using 5-parameter logistic regression in SoftMax Pro version 7.1 and concentrations in the sample extracts were determined by back-calculating the values obtained in the ELISA using this calibration curve and correction for the dilution. Concentrations of ABA-1 in stool were expressed as ng ABA-1 / g stool by multiplying the concentration in the 5-fold diluted extract (in ng/mL) by 20 (based on an estimated 250 mg in 1 mL of extraction buffer).A. lumbricoides DNA in stool, linear regression analysis was performed on log-transformed data. For determination of the association between ABA-1 positive stool and a specific infection, contingency tables were prepared, Fisher’s exact test was performed, and Odds ratio was calculated. For association with A. lumbricoides, all data were used, for association with any other infection, only data from A. lumbricoides negative subjects were used. All analyses were performed using GraphPad Prism version 7.00.Calibration curves for ABA-1 were generated using 5-Parameter Logistic regression and ABA-1 concentration in unknown and calibration samples were back-calculated using this calibration curve. Accuracy and precision were determined by analyzing 4 calibration curves on 3 consecutive days . Accuracy at each calibration point was determined by dividing the average back-calculated value by the nominal value. For each calibration point, the within-run coefficient of variability (CV) was calculated by dividing the average standard deviation (sd) of the three days by the average concentration for that point. The between-run CV was calculated as the sd of the average of each day divided by the average concentration for that point. For evaluation of the correlation between ABA-1 coproantigen ELISA and qPCR-based detection of in vivo by the worm, pseudocoelomic fluid of A. suum and a protein extract of adult A. suum worms were tested in the ELISA , while a weak association was found with T. trichiura infection (P = 0.046). To assess whether possible cross-reaction with a T. trichiura protein was causing this, blast analysis was performed on the T. trichiura proteome, but no homologs could be found . Odds ratio for a positive ABA-1 result was 219 (95% CI: 84.7–540) for A. lumbricoides infection, while this was 4.61 (95% CI: 1.30–16.4) for T. trichiura, 2.61 (95% CI: 0.224–16.2) for hookworm and 0.501 (95% CI: 0.113–2.07) for S. mansoni. Furthermore, there was a highly significant positive correlation between A. lumbricoides DNA copies, and ABA-1 levels found in stool , we used this correlation to determine a morbidity cut-off for ABA-1. Based on the linear regression between ABA-1 levels and DNA copies in stool, a morbidity cut-off for ABA-1 of 18.2 ng/g stool was determined, being the minimal ABA-1 concentration that corresponds to M&HI infection. Of the 71 A. lumbricoides infected individuals studied here, 19 were found to have a M&HI infection based on qPCR. Of those, 13 had ABA-1 levels above this cut-off, corresponding to a sensitivity of 68.4%. Of the 455 samples with no or low intensity infection, only 13 were found to have ABA-1 levels above this cut-off, corresponding to a specificity of 97.1%. Interestingly, 12 false positives had low intensity infection, and only one was negative for A. lumbricoides, reconfirming the high specificity of the ABA-1 ELISA.Since A. lumbricoides infection, stool samples were analyzed from 22 patients with A. lumbricoides infection who were treated with albendazole. Samples were collected at 0, 6, 12 and 24 days after treatment. qPCR analysis on these stool samples showed complete disappearance of STH DNA in all subjects, indicating killing or expulsion of the parasite (A. lumbricoides infection.To further establish the role of ABA-1 as a biomarker for parasite . The quaparasite . These rA. suum is a good model to study A. lumbricoides infection [A. suum eggs/day (low trickle) and 10 pigs were experimentally inoculated with 100 A. suum eggs/day (high trickle). In all collected stool samples, both fecal egg counts (FECs) and ABA-1 levels were determined , it was expected that the antibodies would cross-react with A. suum ABA-1. It was found that at day 42 of the infection, the first pigs started to shed eggs in their stool and similarly, the first ABA-1 positive stool samples were detected at day 42. Two weeks later, already 50% of the pigs were ABA-1 positive, while 60% were positive by Mini-FLOTAC, indicating that ABA-1 appearance is closely related to the appearance of eggs in stool. This latter observation, together with the observation that bead beating was required to release ABA-1 from stool for ELISA testing, might support the hypothesis that ABA-1 in stool is mainly associated with parasite eggs or egg fragments. The concurrent appearance of ABA-1 and eggs in stool of A. suum infected pigs is different to what was observed with the Trichuris vulpis porin coproantigen assay where coproantigen was detected as early as 23 days post-infection while eggs were not observed before day 69 [Additional demonstration of the role of ABA-1 as biomarker for A. lumbricoides infection in that community. Integration of school-based surveillance for STH in the deworming programs or other NTD surveillance programs, might be a very cost-effective approach [Mycobacterium tuberculosis and even plant cell walls [M. tuberculosis antigens in sputum samples, for the circulating cathodic antigen (CCA) in urine as diagnostic test for S. mansoni, and many more [A. lumbricoides, there might also be other proteins with an even better sensitivity/specificity profile.The biggest advantage of a coproantigen test could be its use at the collection site. When school-based deworming programs are being executed, the teams on the ground could use such a test to immediately assess the prevalence and intensity of approach , 41. Tarapproach . It willapproach , 43. To ll walls , 45. Thell walls . The secany more –49. AlsoAscaris ABA-1 in stool sample extracts. Using this ELISA, we demonstrated that ABA-1 in stool is a highly sensitive and specific biomarker to detect infection with A. lumbricoides with ABA-1 levels correlating to infection intensity. A user-friendly and field-adjusted method of this assay could thus potentially be useful in monitoring infection prevalence and intensities in communities during STH control programs.In conclusion, we developed a new sensitive coproantigen ELISA that detects S1 Fig(TIF)Click here for additional data file.S2 FigA total of 2.2 μg of purified protein was loaded and visualized by Coomassie blue staining.(TIF)Click here for additional data file.S3 FigStool samples were collected before (Day 0) and at different timepoints after treatment with albendazole: 6 days, 12 days and 24 days after treatment.(TIF)Click here for additional data file.S1 TableCalibration curves were analyzed in 4-fold over 3 different days and back-calculated concentrations were used to determine accuracy and coefficients of variation.(DOCX)Click here for additional data file.S1 Data(TXT)Click here for additional data file.


One of the key benefits of sol-gel-derived glasses is the presence of a mesoporous structure and the resulting increase in surface area. This enhancement in textural properties has a significant effect on the physicochemical properties of the materials. In this context the aim of this study was to investigate how sol-gel synthesis parameters can influence the textural and structural properties of mesoporous silicate glasses. We report the synthesis and characterization of metal ion doped sol-gel derived glasses with different dopants in the presence or absence of a surfactant (Pluronic P123) used as structure-directing templating agent. Characterization was done by several methods. Using a structure directing agent led to larger surface areas and highly ordered mesoporous structures. The chemical structure of the non-ordered glasses was modified to a larger extent than the one of the ordered glasses due to increased incorporation of dopant ions into the glass network. The results will help to further understand how the properties of sol-gel glasses can be controlled by incorporation of metal dopants, in conjunction with control over the textural properties, and will be important to optimize the properties of sol-gel glasses for specific applications, e.g., drug delivery, bone regeneration, wound healing, and antibacterial materials.  Mesoporous materials (MMs) are defined as materials having pores between 2 to 50 nm according to IUPAC  . They arThe benefits of mesoporous structures include i) the possibility of drug loading and controlled release from the mesopores, and ii) the ability to achieve consistent and controlled degradation due to the ordered and uniform structures obtained using structure-directing agents. These properties are important for biomedical applications  of the mesoporous bioactive glasses. Ideally due to their ordered pore structure, ordered mesoporous glasses could be more effective in their bioactive behavior and drug delivery capability compared to non-ordered glasses. However, if doped with suitable therapeutic elements in the right amount, the non-ordered glasses can also become attractive for antibacterial applications, wound healing, and bone regeneration. Indeed mesoporous bioactive glasses doped with metal elements can degrade homogeneously, thus the release of the therapeutic metal ions can be controlled. Therefore, control of the mesoporosity characteristics plays an important role regarding the mentioned medical applications of mesoporous bioactive glasses.The preparation of mesoporous silica and mesoporous silicate glasses by sol-gel method has been extensively studied ,9,10,11.Mesoporous silicate glasses (MSGs) synthesized with surfactants have enhanced surface area and higher pore volume and in the context of biomedical applications have an increased ability to induce in vitro apatite mineralization in simulated body fluid (SBF), whilst also having excellent cytocompatibility ,14,15,162O, CuO, ZnO, and CeO2. These oxides were chosen for their proven antibacterial properties  were prepared by the sol–gel method. In the first step of undoped-MSG preparation, 50 mL TEOS was mixed with 33.12 mL of distilled water. As a catalyst 6.80 mL 0.5 M HCl was also added and mixed for 30 min. The preparation of doped-MSGs required 46.80 mL of TEOS. The catalyst used for Ag, Zn, and Ce-MSGs was 24.04 mL of 0.5 M HNO3 and for Cu-MSGs 17.50 mL of 0.5 M HCl. In the next step, 4.76 mL TEP was added to the solution and 30 min mixing was done. Afterwards, 9.87 g of Ca(NO3)2·4H2O was added to the solution and stirred for 60 min. For the doped-MSGs, the nitrates of the dopants were added to the sol and stirred for a further of 60 min. The resultant sols were aged for 3 days under a fume hood without stirring, and the gels were dried at 40 °C for 48 h. In the last step, to obtain undoped, Cu, Zn, and Ce-MSGs, heat treatment at 700 °C with 233.33 °C/h heating rate for 3 h was applied. For the Ag-MSGs, the heat treatment was carried out at 500 °C with a 166.66 °C/h heating rate for 5 h. Afterwards, the glasses were left for slow cooling in the furnace.Silica-based undoped and Ag3 was used as the catalyst for Ag, Zn, and Ce-MSGs. After P123 dissolution, 7.13 mL TEOS was added to the solution and stirred for 30 min for undoped-MSGs. Doped-MSGs required 6.65 mL of TEOS. In the next step, 0.67 mL of TEP was added and stirred for a further 30 min. A total of 1.40 g of Ca(NO3)2·4H2O was added and stirred for 1 day for undoped-MSGs. For the doped-MSGs, after stirring 1.40 g of Ca(NO3)2·4H2O for 1 h, the nitrates of the dopants were added to the sol and stirred for 1 day. In the next step, these sols went through evaporation induced self-assembly (EISA) process, by leaving the sols in petri dishes under a fume hood. Finally, heat treatment was done at 700 °C at a 233.33 °C/h heating rate for 3 h in the case of undoped, Cu-MSG, Zn-MSG, and Ce-MSG synthesis. Ag-MSGs were obtained at 500 °C at a 166.66 °C/h heating rate for 5 h. Afterwards, the glasses were left for slow cooling in the furnace.MSGs with ordered mesoporosity were synthesized by evaporation induced self-assembly (EISA)  process High resolution transmission electron microscopy (HRTEM) studies were performed with a FEI Talos F200S microscope  equipped with Super-X energy dispersive X-ray spectrometry (EDX) system, including two silicon drift detectors. The microscope operates at an accelerating voltage of 200 kV. Prior to the HRTEM analyses, MSG powders were deposited on a Holey carbon film on a copper grid. The pore profiles were obtained by using ImageJ analysis to observe the distance between the pore channels. Twenty-five to 150 pore size measurements were done on the MSG samples. The standard deviations of these measurements are also given.The mesoporous properties  of the undoped and doped-MSGs were investigated by nitrogen adsorption porosimetry. The machine used was TriStar II Plus 2.03 . Before performing the adsorption analyses, MSGs were degassed under vacuum for 5 h at 423 K. The analyses were performed at 77 K. Brunauer-Emmett-Teller (BET) method was used to determine the surface area of the MSGs. Barrett-Joyner-Halenda (BJH) method was applied to determine the pore size diameter of the MSGs from the desorption branch of the isotherm.−1) of a GladiATR Vision , attenuated total reflectance accessory that was installed in the sample compartment of an Agilent 660-IR FTIR spectrometer . A press was used to secure the samples in place and to ensure that a sufficient homogeneous contact between the sample and attenuated total reflectance (ATR) crystal was achieved. The samples were measured in the region 4000–400 cm−1 wavenumbers and 32 scans were collected at a resolution of 4 cm−1.Undoped and doped-MSGs were placed directly onto the diamond crystal  method was applied. Axios wavelength dispersive (WD) XRF spectrometer  was used.The X-ray diffraction (XRD) analyses of the MSGs were done by D8 Advance , with Copper Kα radiation at 40 kV and 30 mA. The diffraction range of the analyses was 5 to 70° 2θ. The step size and the dwell time used to collect the data using Bragg-Brentano geometry was 0.02° 2θ and 0.5 s per step, respectively. X-rays were detected simultaneously by a Lynxeye solid state detector over a 3° 2θ window.In contrast, The average pore diameters of the ordered MSGs are shown in 2 adsorption-desorption isotherms of non-ordered and ordered, undoped-MSGs, both the non-ordered and ordered undoped-MSGs show type IV isotherms  for the non-ordered mesoporous glasses can be seen in −1, which can be assigned to the asymmetric Si-O-Si stretching mode .,23.22,23Compared to non-ordered MSGs, the formation of Si-O-NBO bonds in the ordered MSGs was not clearly evident. This can be related to the lower level of dopant incorporation into the glass network as confirmed by XRF analysis . In geneIt was seen from the semi-quantitative XRF results, that the dopant ions were not fully incorporated into the structures of ordered MSGs . Non-ionIn comparison to ordered MSGs, non-ordered MSGs had better dopant incorporation . The reaAll non-ordered MSGs exhibit amorphous behavior with a halo between 15° and 40° 2θ characteristic of amorphous silicate glasses, as seen in 2O in the glass structure can reduce to Ag in the presence of UV light. It has been shown that the addition of silver in bioactive glasses can cause an increase of crystallization of quartz and metallic silver [+ ions have been shown to reduce to Ag [+ reduction into metallic silver. Similarly, Cu2+ ions present in the sol were not completely incorporated into the gel network (as given by XRF data, 2-15CaO-5P2O5 BG synthesized via P123 assisted sol-gel process [Among the ordered MSGs, 5 wt% Ag and Cu-MSGs showed silver and copper oxide peaks in their XRD diffractograms b; 5 wt% c silver . In ethace to Ag . In the  process . It was MSGs were synthesized by sol-gel method with and without a surfactant to obtain ordered and non-ordered mesoporosity, respectively. These MSGs were compared in terms of their structural properties. MSGs with highly ordered mesoporosity were obtained by the surfactant-assisted synthesis route and these MSGs exhibited larger surface areas and higher pore volumes than the non-surfactant assisted MSGs. FTIR spectra showed that the addition of dopant ions to non-ordered MSGs modified the glass network more than was the case with ordered MSGs due to a higher extent of dopant incorporation into the product glass structures. This was further demonstrated by XRF analysis. The addition of copper to both non-ordered and ordered MSGs was shown to decrease the surface area the most and it changed the position of the Si-O-Si vibration peaks in FTIR. The developed MSGs should be characterized further for example using solid state NMR, computer simulation, X-ray scattering experiments, Raman spectroscopy or XPS analysis and, in particular, such advanced characterization may help to explain the observed increase in textural properties for Ce-MSGs.


The results showed that both plantation age and eucalyptus species could affect the total microbial biomass and fungal biomass, whereas the bacterial biomass was affected only by plantation age. The fungal biomass and the fungi-to-bacteria ratio significantly increased along with increasing plantation age. Similarly, the plantation age and eucalyptus species significantly affected the enzyme activities associated with carbon cycling . The activities of β-d-glucuronidase and β-glucosidase were significantly higher in the E. camaldulens plantation. The enzymes involved in nitrogen (N-acetyl-glucosamidase) and sulfur (sulfatase) cycling were only affected by the eucalyptus plantation age and species, respectively. The results highlight the importance of the age and species of eucalyptus plantations on soil microbial activities.Soil microorganisms and extracellular enzymes play important roles in soil nutrient cycling. Currently, China has the second-largest area of eucalyptus plantations in the world. Information on the effects of eucalyptus age and species of trees on soil microbial biomass and enzyme activities, however, is limited. In this paper, the soil microbial biomass and enzyme activities were studied in eucalyptus plantations with different ages (1 and 5 Eucalyptus grandis than that of slash pine [Soil microbes play a vital role in soil quality and function due to their ability to participate in the degradation of soil organic matter . Their aash pine . Moreoveash pine . TherefoCunninghamia lanceolate) to cauliflower shell (Mytilaria laosensis) due to improved litter quality [Camellia sinensis) plantation aged 50 had a significantly higher microbial biomass compared to that aged nine in Zhejiang province, China [The soil microbial biomass is a measure of the mass of the living component of soil organic matter. It is widely considered as the index of soil fertility and ecosystem productivity . In fore quality . Compare quality . Moreove quality . For exa quality . The teae, China . Even a e, China . Therefoe, China .Myrtaceae, is originally from Australia and nearby islands, and is considered worldwide as the fast-growing genera of trees [2 until the mid-1980s [E. grandis can decrease the soil nutrient content in the plantation in the Rio Doce Valley region [E. tereticornis planting can improve the soil nutrient levels, including the soil organic C, total N and available P, and the exchange capacity for Ca+ and Mg+ [E. urophylla×E. grandis and E. camaldulens are the two main afforestation tree species in South China [E. pellita, but little is known about its adaptation [E. urophylla×E. grandis, E. camaldulens and E. pellita) and ages  were selected to: (1) investigate the effect of plantation age and species on the total soil microbial biomass and fungal and bacterial biomass, and (2) evaluate the response of the extracellular enzyme activities involved in nutrient cycling to different plantation ages and species.Eucalyptus, belonging to the genera of of trees . The extid-1980s . The shoid-1980s ,25. A pry region . With in and Mg+ . Nowaday and Mg+ ,29. E. uth China . In addith China . Anotheraptation . InformaThe study site was located in the research station of the China Eucalypt Research Centre (CERC), Chinese Academy of Forestry, Zhanjiang, China, which belongs to the Northern Leizhou Peninsula, Southern China (latitude 21°27′ and longitude 110°11′ E). The study site was in the northern part of the Leiqiong island which has a maritime monsoon climate . The soiE. urophylla × E. grandis (EUG), E. camaldulensis (EC) and E. pellita (EP). Each of the tree species had a one-year plantation (1-year) and a five-year plantation (5-year), except for EP which had an 11-year rather than a five-year plantation. Therefore, 5+-year is set to refer to the five-year and 11-year plantations. The 1- and 5+-year plantations were approximately 500 m apart. Three 15 m × 15 m plots with a 20 m distance were selected in each plantation. Three trees were chosen randomly in each plot for soil collection. The distance between the trees in each plantation was 3 m × 2 m. The soil samples were collected at 0–10 cm soil after litter removal at a 1 m distance from the tree with three directions at a 120°. The three subsamples from each tree were mixed together as one sample. In total, 54 samples were obtained (9 samples per plantation). The soil samples were transported to the laboratory on ice, where the samples were further divided and stored for soil enzyme activity and microbial biomass analysis (stored at +4 °C) and DNA isolation (stored at −20 °C).The soil samples were collected on 18 May 2017. Six plantations were chosen and each had an area of 120 m × 180 m. The selected plantations included two plantation ages and three eucalyptus species of tree. The three selected species were 2SO4 solution , respectively, in an oscillator and shaken for 30 min. The suspension was then vacuum filtered with a 0.45 μm polycarbonate filter membrane. The soil microbial biomass carbon in the filtrates was determined by the wet-oxidation method with K2Cr2O7 [The chloroform fumigation extraction method was used to measure the soil microbial biomass . Briefly K2Cr2O7 .A quantity of 0.3g (fresh weight) of homogenized soil was used to extract the total genomic DNA using the Soil DNA Kit  according to the manufacturer’s instructions. The concentration of the extracted DNA was measured by a Nanodrop-1000 spectrometer .The absolute amount of fungi and bacteria in the soil samples was detected by real-time quantitative PCR (qPCR). The specific-primer pair FF390 (5′-ATTACCGCGGCTGCTGG-3′) and FR1 (5′-AIC-CATTCAATCGGTAIT-3′) were used to amplify the fungal 18S ribosomal RNA (rRNA) genes , and 338Phlebia radiata FBCC43 (genome size 40.92 Mb) for fungi and Escherichia coli H673 for bacteria  as the copy number of the 18S rRNA gene and the 16S rRNA gene were known [−1 and calculated by the following equation:r is the preset concentration of the template DNA; Vr is the volume of template DNA added into the PCR amplification system; and m indicates the dry weight of the soil used to extract the template DNA.The standard curves were conducted by using DNA extracted from −1·h−1).Seven soil enzyme activities related to the carbon cycle (β-xylosidase (XYL), β-d-glucuronidase (GLR), β-cellobiosidase (CEL) and β-glucosidase (GLU)), nitrogen cycle (N-acetyl-glucosamidase (NAG)), phosphorus cycle (phosphatase (PHO)) and sulfur cycle (sulfatase (SUL)) were analyzed. The enzyme activity was measured fluorometrically with 96-well plates by using 4-methylumbelliferone-linked (4-MUB) enzyme substrates . In shorThe differences in microbial biomass and enzyme activities between samples were assessed by one-way analysis of variance (ANOVA) by Duncan’s tests. The effects of the interaction between eucalyptus species and age on the measured parameters were tested by two-way ANOVA. All the analysis was done by SPSS v16.0 . Additionally, the correlations between the enzyme activity or microbial biomass and the soil’s physio-chemical properties, which were cited as p < 0.05, p < 0.05, p < 0.05, p < 0.05, For the eucalyptus plantations with different ages, the soil fungal and bacterial biomasses, and the ratio of fungal and bacterial biomass (F:B) differed  and S (SUL) cycles differed between the 1-year and 5p < 0.05, p < 0.05, +-year plantations (p < 0.05) compared to the EUG and EP plantations. The β-cellobiosidase activity was distinctly lower in the EP plantation than in the EUG and EC plantations (p < 0.05). The β-d-glucuronidase activity in the EC plantation was significantly higher compared to the EUG and EP plantations. In the 5+-year plantations, the β-d-glucuronidase activity and N-acetyl-glucosamidase activity were significantly lower in the EUG plantation than that in the EP and EC plantations, respectively (p < 0.05). The sulfatase activity was significantly higher in the EC plantation than that in the EUG and EP plantations (p < 0.05). The two-way ANOVA showed that eucalyptus age and species had a distinct impact on the enzyme activities, with the exception of phosphatase , whereas the activity of β-xylosidase was negatively correlated to the C:N ratio . The NAGThe soil microbial biomass, fungal biomass and F:B ratio varied significantly among plantations with different species and ages of eucalyptus trees. The changes in substrate and the availability caused by tree species were the main factors to determine the microbial abundance . The soiFungi are the most important decomposer of litter and plant tissue, whereas bacteria are more sensitive to soil changes than fungi . NitrogeThe soil enzyme activities differed among eucalyptus tree species. The tree species are associated with unique microbial communities, which strongly affect soil enzyme activities ,56. The N-acetyl-glucosamidase (NAG) is involved in the decomposition of chitin and the fungal cell wall . N additThe sulfatase activity significantly increased along with the plantation age in our study. Sulfatase is involved in the hydrolyzation of sulfate esters and is commonly secreted by bacteria when sulfur availability is limited . The actThe soil microbial biomass and fungal biomass differed among eucalyptus species. With increasing plantation age, the fungal biomass, bacterial biomass and F:B ratio significantly increased. Similarly, the plantation age and species significantly affected the enzyme activities associated with carbon cycling. The enzyme involved in sulfur cycling (S) was strongly affected by the eucalyptus species, whereas the enzyme involved in nitrogen (N) cycling increased with plantation ageing and was affected mainly by the eucalyptus plantation age. In addition, the soil’s physio-chemical properties, as caused by eucalyptus species and plantation age, can also affect the soil microbial biomass and enzyme activities indirectly. The results highlight the importance of the age and species of eucalyptus plantations on soil microbial activities.


We aimed to assess the effect of sulforaphane (SFN) on breast cancer cell migration and also its effect on the expression of epithelial mesenchymal transition (EMT) markers and β-catenin.This study was performed in Shahroud University of Medical Sciences, Shahroud, Iran from 2017–2018. In this experimental study, MDA-MB-231 cells were treated with different concentrations of SFN  at different time points of 24, 48, and 72 h. The control group was untreated cells. The inhibitory effects of different concentrations of SFN on cell migration at different time points were evaluated using scratch assay. Moreover, apoptosis was assessed by using flow cytometric analysis. The expression of β-catenin and EMT markers of ZEB1, fibronectin, and claudin-1 were determined by real-time PCR. Western blotting analysis of β-catenin was applied to determine its changes after SFN treatment.SFN markedly inhibited the migration of cells at concentrations of 10, 20, 30, and 40μM after 24, 48, and 72 h. At relatively, high concentrations , SFN induced apoptosis. Moreover, SFN reduced the gene expression of ZEB1, fibronectin, and claudin-1 after 72 h. The expression of β-catenin revealed a time-dependent decrease at the concentration of 40 μM SFN.Downregulation of EMT markers and β-catenin showed accordance with the inhibition of migration. SFN could be a promising drug candidate to reduce metastasis in breast cancer. Breast cancer is the most common cancer in women and the leading cause of cancer-related death among females worldwide. In fact, the cause of death in many patients with breast cancer is tumor spreading to other parts of body. Currently, there is not a cure for metastatic breast cancer and patients live approximately five years after initial diagnosis .Metastasis is an enormously complex biological process involving different genes and biomolecules. More recently, epithelial-mesenchymal transition (EMT) has been shown to be one of the key regulators of cancer metastasis. EMT is a physiological process by which epithelial cells lose their adherent junctions and apical-basal cell polarity to form spindle-shaped cells that contribute to their ability to migrate as single cells. Loss of epithelial markers such as E-cadherin and acquisition of mesenchymal markers like fibronectin is a fundamental event in EMT. This switch in cell structure and behavior is mediated by key transcription repressors such as zinc finger proteins of ZEB family . AdditioIn recent years, much attention has been directed towards therapeutic strategies based on targeting β-catenin and EMT markers as the key players in cancer metastasis. There is a constant demand to develop less toxic, more efficacious, and affordable anticancer drugs with reduced side effects. In recent years, cancer prevention by natural products has received considerable attention. Among vConsidering the promising anticancer properties of SFN, the aim of this study was to evaluate the effects of various concentrations of SFN on cell migration in MDA-MB-231 human metastatic breast cancer cells at different time points of 24, 48, and 72 h. Moreover, the expression of certain key elements of EMT, including ZEB1, fibronectin, and claudin-1 in breast cancer cells were examined in vitro after treatment with SFN. Furthermore, as upregulation of the Wnt/β-catenin signaling pathway has also been shown to lead to tumor metastasis, our present study was designed to determine the expression level ofβ-catenin in MDA-MB-231 breast cancer cells in response to SFN.In this in vitro experimental study, human breast cancer cell line (MDA-MB-231), was obtained from the Pasteur Institute, National Cell Bank of Iran. The study was performed in Shahroud University of Medical Sciences, Shahroud, Iran from 2017–2018. The SFN was purchased from Sigma Company. Cells were cultured in Dulbecco modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (FCS), and antibiotics . Cells were incubated at 37 °C in a humidified atmosphere composed of 95% air and 5% CO2.5 cells/well in six-well plates. Cells were treated with different concentrations of SFN . Untreated cells were considered as control group. After time points of 24, 48, and 72 h, the cells were trypsinized and washed with PBS. Annexin-V-FITC/PI labeling was performed according to the manufacturers’ instructions. Quantification of Annexin-V/propidium iodide incorporation was performed using a FACScalibur flow cytometer . Acquired data were analyzed using the Win-MDI software.MDA-MB-231 cells were plated at a density of 2×10The effect of SFN treatment on cell migration was determined using scratch assay as described previously . BrieflyBy using TriPure isolation reagent  based on the manufacturer’s instructions, total RNA was extracted from cells. The extracted RNAs were treated with DNase I enzyme to eliminate probable contamination with genomic DNA. Reverse transcription and real-time PCR were performed following the manufacturer’s instructions using SYBR Green Master Mix . Primers used in this study are shown in The pellets of control and treated cells after the proposed time points of 24, 48, and 72 h were suspended in lysis buffer , supplemented with protease inhibitor cocktail (Sigma-Aldrich). Forty micrograms of proteins were denatured using Laemmli’s sample Buffer and boiled at 95 °C for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Amersham). The membranes were placed in a blocking buffer for 60 min at RT and subsequently incubated with anti-β-catenin primary antibody  overnight, at 4 °C. The following day, the membranes were washed and incubated with a peroxidase conjugate secondary antibody  for 60 min, at RT. Then membranes were washed and incubated with the Bio-Rad Clarity™ western ECL substrate and finally exposed to X-ray film from FUJIFILM Corporation. Band intensity was calculated using Image J software.The statistical analyses were performed using GraphPad Prism version 6.0 software. One way ANOVA with Dunnett's multiple comparison test was used to assess differences between various groups. Data are presented as mean ± standard deviation (SD).To investigate the effect of SFN on cell metastatic potential, the migration capacity was detected using scratch assay. SFN suppressed migration of cells to the denuded zone at concentrations of 10, 20, 30, and 40 μM . These fThe ability of SFN to induce apoptosis was assessed by flow cytometry analysis. MDA-MB-231 cells were treated with different concentrations of SFN  for 24, 48 and 72 h. As shown in We performed quantitative RT-PCR to examine the effect of SFN on expression levels of specific genes involved in EMT such as ZEB1, fibronectin and claudin-1. In the present study, MDA-MB-231 cells were treated with different concentrations of SFN . Total RNAs were collected at 24, 48 and 72 h. We found that SFN significantly downregulated the expression of ZEB1 at 40μM after 72 h compared with the control group. SFN also decreased the expression of fibronection at 20, 30, and 40 μM after 72 h. Decreased expression of claudin-1 was observed at 30 and 40μM after 72 h .To further investigate the possible mechanisms by which SFN exerts its anti-migratory effect, we evaluated whether the expression of β-catenin is associated with SFN treatment. Immunoblot analysis was employed to examine the expression of β-catenin. As shown in The present study illustrates that SFN exposure induces apoptosis at 30–40μMand also inhibits the migratory capabilities of MDA-MB-231 cells at 10–40μM. These results raise the possibility that SFN may be a promising candidate to prevent breast cancer progression and metastasis. It was revealed that the anti-cancer activity of SFN is modulated by a variety of cell-signaling pathways including NF-κB signaling , Keap1– EMT is a crucial step for tumor cell migration and invasion in various types of human cancers. Overexpression of ZEB1, as a transcription factor, is critical for induction of EMT, 16. TheFibronectin is another established EMT marker, which appears to be involved in extracellular matrix (ECM) remodeling during EMT. It is upregulated during development and has been linked to the growth, survival and metastasis in variety of cancers including breast , prostatMoreover, claudins, the integral membrane proteins which form the backbone of tight junctions, are aberrantly expressed in diverse types of human cancers . Claudinthere is considerable interest in developing Wnt/β-catenin pathway inhibitors as anti-cancer therapeutics  have been completely observed by the authors.